Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

Department of Biological Sciences, University of North Texas, Denton, Texas 76203, USA.

Abstract

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

Nitrate uptake in X. laevis oocytes expressing MtNIP/LATD. Oocytes were microinjected with MtNIP/LATD mRNA (black bars, +) or water as a negative control (gray bars, −), incubated for 3 d, and then placed for the indicated times in medium containing 250 μm or 5 mm NO3− at pH 5.5 or 7.4. The oocytes were rinsed, lysed, and assayed for NO3− content. A and C, Treatment with 250 μm NO3−. B and D, Treatment with 5 mm NO3−. A and B, pH = 5.5. C and D, pH = 7.4. Data are shown for one biological replicate ± sd (n = 3–5 batches of 4–6 oocytes per batch). Asterisks mark NO3− uptake significantly different from the negative control, using Student’s t test at P < 0.05. Similar results were obtained in more than five repetitions of the experiment. E, Michaelis-Menten plot of oocyte NO3− uptake. MtNIP/LATD-injected oocytes (squares) or water-injected oocytes (circles) as control were incubated for 3 h in 50 μm to 10 mm NO3− in batches of five and assayed for NO3− uptake. Results for two biological replicates are indicated by the black and gray symbols, with error bars showing sd. All NO3− uptake was significantly different from the negative control, using Student’s t test at P < 0.05, except for that at 50 μm. F, Hanes-Woolf plot of averaged NO3− uptake data, in MtNIP/LATD-injected oocytes minus water-injected oocytes, presented in E. These data were used to calculate the Km of 160 μm.

Nitrate uptake in X. laevis oocytes expressing MtNIP/LATD or mutant Mtnip/latd mRNAs. Oocytes were microinjected with MtNIP/LATD mRNA, mutant mRNA, or water as a negative control, incubated for 3 d, and then placed for the indicated times in medium containing 5 mm or 250 μm nitrate, at pH 5.5, and assayed for nitrate uptake. A, Treatment with 5 mm nitrate. B, Treatment with 250 μm nitrate. C, Treatment with 5 mm nitrate. Oocytes expressing wild-type (WT) MtNIP/LATD (black bars), Mtnip-1 (dark gray bars), Mtnip-3 (horizontally striped bars), Mtlatd (hatched bars), or water as a negative control (gray bars) are shown. Data are shown for one biological replicate ± sd (n = 3–5 batches of 4–6 oocytes per batch). Asterisks mark nitrate uptake that is significantly different from the negative control, using Student’s t test at P < 0.05. Similar results were obtained in more than three repetitions of the experiment.

MtNIP/LATD complements the chlorate-insensitivity phenotype of the Arabidopsis chl1-5 mutant. Arabidopsis chl1-5 plants were transformed with a construct containing MtNIP/LATD under the control of the Arabidopsis EF1α promoter, pAtEF1α-MtNIP/LATD, or a positive control construct containing the Arabidopsis AtNRT1.1 gene under the control of the same promoter, pAtEF1α-AtNRT1.1. The plants were treated with chlorate, a NO3− analog that can be converted to toxic chlorite after uptake, as described by . A, Arabidopsis plant. B, Arabidopsis chl1-5 plant. C, Arabidopsis chl1-5/pAtEF1α-AtNRT1.1 plant. D, Arabidopsis chl1-5/pAtEF1α-MtNIP/LATD plant. Bars = one-quarter inch. The MtNIP/LATD gene was able to confer chlorate sensitivity on Arabidopsis chl1-5 plants, similar to the AtNRT1.1 gene.